Part:BBa_K660004:Experience
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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:
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.
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.
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
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