Difference between revisions of "Part:BBa K660004"

Revision as of 17:34, 14 October 2014

iLOV

iLOV is a a variant of LOV2 (https://parts.igem.org/Part:BBa_K660000) which has been isolated over time as it shows better fluorescence and photo-stabiltility.

For your convenience, a separate BioBrick (BBa_K660003) has been created which is already ligated to a strong RBS and double terminator, making iLOV the ideal reporter for characterisation of new promoters. The page for this Biobrick is located here: (https://parts.igem.org/Part:BBa_K660003).

In studies whereby it has been used in comparison to GFP to track plant infections, it has outperformed GFP. It is much smaller than GFP at around 300 bp as opposed to 700 bp, and for this reason it better suited as a reporter for the movement of things such as viruses, and tagging of small proteins. Unlike GFP which photo-bleaches irreversibly, iLOV undergoes spontaneous recovery from photo-bleaching under high intensity exposure to UV. This photo-bleaching reversibility is due to the changing state between the fluorescent and non-fluorescent form of the bound FMN chromopore

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Figure 1: Digram showing movement of TMV (tobacco mosaic virus). On the left shows TMV with iLOV, and the centre and right show TMV with GFP. We can see that TMViLOV shows systemic infection, whereas TMVGFP shows poor, or no infection.Image taken from: Chapman, S. et al (2008) The photo-reversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. PNAS, 105 (50) pp. 20038 - 20043

(Full article available at: http://www.pnas.org/content/105/50/20038.full)

Figure 2: Image demonstrating photo-bleaching reversibility of iLOV. Left – iLOV pre-bleach, Centre – iLOV post-bleach, and Right – iLOV post-recovery. Image taken from Chapman et al (2008) The photo-reversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. PNAS, 105 (50) pp. 20038 - 20043

(Full article available at: http://www.pnas.org/content/105/50/20038.full)

Another advantage of LOV in comparison to other fluorescent proteins is it's ability to work in anoxic conditions, making it ideally suited to work within biofilms. Fluorescent imaging of iLOV can be done by using an excitation wavelength of 476nm, with fluorescent emission between 510 and 550 nm. (Chapman et al 2008)

This protein has been codon optimised for E.coli expression.

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).

(Full article available at: http://2014.igem.org/Team:Exeter/iLOVCharacterisation)

Sequence and Features