Difference between revisions of "Part:BBa K660004:Experience"

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Applications of BBa_K660004

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UNIQdb67b3caf3960f37-partinfo-00000000-QINU

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Exeter University 2014

As part of the Exeter 2014 project iLOV was used as a reporter gene in two possible biosensors. iLOV was used to test the effectiveness of a NemR upstream intergenic region (https://parts.igem.org/Part:BBa_K1398005) in part BBa_1398004 (https://parts.igem.org/Part:BBa_K1398004). Part BBa_1398007 (https://parts.igem.org/Part:BBa_K1398007) used iLOV to test the effectiveness of a NemR recognizing promoter, part BBa_1398008 (https://parts.igem.org/Part:BBa_1398008).

Before we used iLOV as a reporter we first had to characterise it within E. coli, as currently within iGEM iLOV is only demonstrated to work within plant cells. We did this in two ways:

Expressing iLOV within E. coli.

First we expressed iLOV constitutively within E. coli. We did this to check if iLOV can actually fluoresce in E. coli, as there is no characterization on the registry confirming this.

Comparative_Fluoresence_of_iLOV_and_GFP_in_E._coli.png

Figure 1: The fluorescence of WT-cultures, as well as cultures expressing GFP and iLOV. The fluorescence of iLOV (at excitation = 440 nm, emission = 520 nm) reaches a 4400 at 20h, while WT cultures at 20h have a fluorescence of 880.

The fluorescence of iLOV in E. coli can clearly be seen in our experiment. There is clearly a huge increase in fluorescence compared to WT E. coli. It can also be seen that under regular conditions maximal iLOV fluorescence is reached between 20-25 hours. This information was used when selecting a time point for the comparison of fluorescence to optical density. The relative fluorescence of GFP and iLOV are not comparable in this experiment as they are under the control of different promoters, although it is suspected that the fluorescence of GFP is greater. Therefore we can confirm that iLOV can be used as reporter gene within E. coli. Further information on topic this is available here: http://2014.igem.org/Team:Exeter/Detection .

Characterizing the Emission/Excitation Topography of iLOV.

Second, as part of our project the fluorescence topography of iLOV was mapped. The fluorescence response of iLOV was greatest when excited with photons of wavelength 449±1nm whilst photon emissions where detected at a wavelength of 494±2nm. Therefore these are the ideal measurement ranges to use when using the iLOV protein

Fluorescence_of_iLOV_at_a_range_of_Excitation-Emission_wavelengths.png

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 <I>Exeter University 2014</I>
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-As part of the Exeter 2014 project iLOV was used as a reporter in two constructs. iLOV was used to test the effectiveness of a NemR upstream intergenic region (https://parts.igem.org/Part:BBa_K1398005) in part BBa_1398004 (https://parts.igem.org/Part:BBa_K1398004). Part BBa_1398007 (https://parts.igem.org/Part:BBa_K1398007) used iLOV to test the effectiveness of a NemR recognizing promoter, part BBa_1398008 (https://parts.igem.org/Part:BBa_1398008).
+As part of the Exeter 2014 project iLOV was used as a reporter gene in two possible biosensors.
+iLOV was used to test the effectiveness of a NemR upstream intergenic region (https://parts.igem.org/Part:BBa_K1398005) in part BBa_1398004 (https://parts.igem.org/Part:BBa_K1398004). Part BBa_1398007 (https://parts.igem.org/Part:BBa_K1398007) used iLOV to test the effectiveness of a NemR recognizing promoter, part BBa_1398008 (https://parts.igem.org/Part:BBa_1398008).
+Before we used iLOV as a reporter we first had to characterise it within ''E. coli'', as currently within iGEM iLOV is only demonstrated to work within plant cells. We did this in two ways:
+'''Expressing iLOV within ''E. coli''.'''
+First we expressed iLOV constitutively within E. coli. We did this to check if iLOV can actually fluoresce in E. coli, as there is no characterization on the registry confirming this.
+https://static.igem.org/mediawiki/parts/2/25/Comparative_Fluoresence_of_iLOV_and_GFP_in_E._coli.png
+''Figure 1: The fluorescence of WT-cultures, as well as cultures expressing GFP and iLOV. The fluorescence of iLOV (at excitation = 440 nm, emission = 520 nm) reaches a 4400 at 20h, while WT cultures at 20h have a fluorescence of 880.''
+The fluorescence of iLOV in E. coli can clearly be seen in our experiment. There is clearly a huge increase in fluorescence compared to WT E. coli. It can also be seen that under regular conditions maximal iLOV fluorescence is reached between 20-25 hours. This information was used when selecting a time point for the comparison of fluorescence to optical density. The relative fluorescence of GFP and iLOV are not comparable in this experiment as they are under the control of different promoters, although it is suspected that the fluorescence of GFP is greater. Therefore we can confirm that iLOV can be used as reporter gene within ''E. coli''.
+Further information on topic this is available here: http://2014.igem.org/Team:Exeter/Detection .
+'''Characterizing the Emission/Excitation Topography of iLOV.'''
+Second, as part of our project the fluorescence topography of iLOV was mapped. The fluorescence response of iLOV was greatest when excited with photons of wavelength 449±1nm whilst photon emissions where detected at a wavelength of 494±2nm. Therefore these are the ideal measurement ranges to use when using the iLOV protein
+https://static.igem.org/mediawiki/parts/d/d4/Fluorescence_of_iLOV_at_a_range_of_Excitation-Emission_wavelengths.png
-(Full article available at:http://2014.igem.org/Team:Exeter/Detection#TheNemRpromoterconstruct%28BBa_K1398007%29)
-As part of their project the fluorescence topography of iLOV was mapped. The response of iLOV to a low power light source was also explored. The fluorescence response of iLOV was greatest when excited with photons of wavelength 449±1nm whilst photon emissions where detected at a wavelength of 494±2nm.
-https://static.igem.org/mediawiki/2014/e/e7/Exeter2014_iLOV_ResizedBroad.png
-(Full article available at: http://2014.igem.org/Team:Exeter/iLOVCharacterisation)
 |};