Part:BBa_K660004:Experience

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Applications of BBa_K660004

Edinburgh_OG Contribution!

This part was retrieved from the iGEM 2016 distribution kit. BBa_K660004, subsequently improved (BBa_K1968016) by making it compatible with the phytobrick standard and was assembled into the PhytoBricks Universal Acceptor (BBa_P10500). The sequence of this part was confirmed by Sanger Sequencing.

This part was also codon optimized for four organisms, E. coli, Penicilium roqueforti, Rhodoccocus justii, and Synechocystis sp. PCC6803. It was optimized based on two different algorithms (BBa_K1968022, BBa_K1968023), and were also made compatible with the phytobrick standard.

User Reviews

UNIQf2fdc468284b973a-partinfo-00000000-QINU

••• 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. Figure 1: The fluorescence of WT-cultures (negative control), as well as cultures expressing GFP (positive control) and iLOV. The fluorescence of iLOV (at excitation = 440 nm, emission = 520 nm) reaches a maximum of 4400 at 20h, while WT-Top10 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. We also attempted to characterise two further properties of iLOV in E. coli: Anoxic Fluorescence. Autorecovery of Fluorescence after photobleaching. These two properties are important as they differentiate it from GFP, which requires molecular oxygen to fluoresce and does not recover from photobleaching. However, we were not able to demonstrate either of these properties within E. coli. Fluorescence did not occur when iLOV-E. coli was grown under anoxic conditions, and iLOV did not demonstrate any noticeable recovery after photobleaching. This may be because these properties are not shown in E. coli, or because of flaws in our methodology. This is definitely an area which could be further explored by future iGEM teams.

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