Subsequently, other families of GFP variants were discovered and GFP mutants were engineered. These families of GFP-like proteins were discovered in a class of non-bioluminescent Anthozoa (corals). These GFP-like variants have differing properties with regards to chromophore, excitation and emission wavelengths (Tsien & Tsien, 1998) e.g. red & later in other coral species - yellow, cyan and orange. The only other species that fluoresces green is Renilla reniformis (Matz et al., 1999). Phylogenetic analysis has suggested that these colour variants have evolved multiple times recently (Labas et al., 2002). The significance of using different coloured GFPs is to allow visualisation of cell movement or migration in real time with no need for fixing
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This was not ideal for its uses in biotechnology and as a result, GFP was engineered/ mutagenized to allow for a wider range of usage in a diverse set of organisms as they have improved brightness and photo-stability (Matz, Lukyanov & Lukyanov, 2002; Ehrenberg, 2008). The GFP mutants also have differing pH and temperature sensitivities (Ward et al., 1980) in contrast to wild-type GFP which is stable and its fluorescing property is tolerant of a range of pHs and temperatures (Hicks, 2002). GFP was found to be highly stable in temperatures up to 65° C (Cubitt et al., 1995). The S65T mutant was found to have the most useful change as it has approximately a six fold greater brightness than wild-type GFP, no photo-isomerization and slow photo bleaching (Cubitt et al., 1995). In a study by (Protein et al., 1998), two GFP colour variants were tagged to two different proteins and were proven successful in double labelling to provide insight into the effects of chemical inhibition in compartment of the cell.
However, the importance of GFP and its potential uses were only made viable for discovery when cDNA of GFP was first cloned in 1992. This progress would allow for expression in other non-GFP expressing organism (Prasher et al., 1992). This was first demonstrated by Chalfie et al.,1994 and Inouye and …show more content…
victoria’s GFP in prokaryotic Escherichia coli and eukaryotic Caenorhabditis elegans cells. The E. coli cells transformed to express the gfp gene, fluoresced green as opposed to the control bacteria. They also showed that GFP had no apparent negative side effect on the living cells as the media contained the inducer IPTG. This was an indication that GFP wold be a great candidate for observing gene expression and subcellular localization of proteins. GFP’s may also be used for identification of chromosome replication and organization, protein-protein interactions, cell division, intracellular transport pathways and organelle inheritance as the only requirement for detecting GFP is UV or blue light and it does not interfere with cell growth. (M Chalfie, Y Tu, G Euskirchen, WW Ward,
This was not ideal for its uses in biotechnology and as a result, GFP was engineered/ mutagenized to allow for a wider range of usage in a diverse set of organisms as they have improved brightness and photo-stability (Matz, Lukyanov & Lukyanov, 2002; Ehrenberg, 2008). The GFP mutants also have differing pH and temperature sensitivities (Ward et al., 1980) in contrast to wild-type GFP which is stable and its fluorescing property is tolerant of a range of pHs and temperatures (Hicks, 2002). GFP was found to be highly stable in temperatures up to 65° C (Cubitt et al., 1995). The S65T mutant was found to have the most useful change as it has approximately a six fold greater brightness than wild-type GFP, no photo-isomerization and slow photo bleaching (Cubitt et al., 1995). In a study by (Protein et al., 1998), two GFP colour variants were tagged to two different proteins and were proven successful in double labelling to provide insight into the effects of chemical inhibition in compartment of the cell.
However, the importance of GFP and its potential uses were only made viable for discovery when cDNA of GFP was first cloned in 1992. This progress would allow for expression in other non-GFP expressing organism (Prasher et al., 1992). This was first demonstrated by Chalfie et al.,1994 and Inouye and …show more content…
victoria’s GFP in prokaryotic Escherichia coli and eukaryotic Caenorhabditis elegans cells. The E. coli cells transformed to express the gfp gene, fluoresced green as opposed to the control bacteria. They also showed that GFP had no apparent negative side effect on the living cells as the media contained the inducer IPTG. This was an indication that GFP wold be a great candidate for observing gene expression and subcellular localization of proteins. GFP’s may also be used for identification of chromosome replication and organization, protein-protein interactions, cell division, intracellular transport pathways and organelle inheritance as the only requirement for detecting GFP is UV or blue light and it does not interfere with cell growth. (M Chalfie, Y Tu, G Euskirchen, WW Ward,