Difference between revisions of "Part:BBa K1055001"
 
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<partinfo>BBa_K1055001 short</partinfo>
 
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LssmOrange is a fluorophor derived from dsRed. It features the excitation maxima at 437 nm and the emission maxima at 571 nm.  
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LssmOrange is a fluorophor derived from dsRed and is a FRET partner of mKate [[https://parts.igem.org/Part:BBa_K1055000 BBa_K1055000]]
  
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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===
  
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[[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''']]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1055001 SequenceAndFeatures</partinfo>
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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.
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[[Image:LSSmOrange_Diagramm.png|center|400px|thumb|Figure 2. '''Excitation spectrum (dashed line) and emission spectrum (solid line) of LSSmOrange with marked maximums''']]
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[[Image:FRET_Normalized_2.png|center|400px|thumb|Figure 3. '''Overlay of exitation spectrum (dashed line) and emission (solid line) of mKate and LSSmOrange''']]
  
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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:
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===Functional Parameters===
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<partinfo>BBa_K1055001 parameters</partinfo>
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'''Characterization LssmOrange'''
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[[Image:LssmOrange_first_activeHelix.png|center|400px|thumb|]]
  
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This biobrick did measure up to our expectations as shown in the following data. We also used this promoter to express our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.(''E. Coli'' membrane anchor protein). AraC-Pbad shows a low background activity and a good respose to the induction with Arabinose.  
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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.
  
  
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[[Image:Spectra_LssmOrange.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
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[[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.''']]
  
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[[Image:MKate-LSSmOrange_FRET_Pair.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
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=== References ===
  
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[[Image:LssmOrange_bac.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
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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
  
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[[Image:Grow_log.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
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http://www.evrogen.com/products/basicFPs.shtml
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1055001 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K1055001 parameters</partinfo>
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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

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:

LssmOrange first activeHelix.png

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]