Information Technology Reference
In-Depth Information
Green Fluorescent Protein
The green fluorescent protein has been isolated and cloned from the jellyfish
Aequorea victoria
. Due to its internal covalently bound imidazolinone chro-
mophore, GFP is autofluorescent, and the addition of cofactors or exogenous
substrates is not required to produce light [74]. The maxima of the GFP ex-
citation spectrum resides at 395 nm with a minor peak at 475 nm, while the
emission maxima occurs at 509 nm with a small shoulder at 540 nm [116].
Although, the fluorescence quantum yield of GFP (0.72-0.85) is comparable to
that of fluorescein (0.91), its lower molar absorptivity leads to an approximately
1 order of magnitude lower intensity than fluorescein [108].
Advantages of GFP include high stability at biological pH and lack of en-
dogenous homologues in most target organisms [25, 74]. However, it has re-
cently been reported that GFP is toxic to some mammalian cell types, and this
cytotoxicity may be linked to apoptosis [63]. Still, the unique characteristics
of GFP have led to its successful application in gene expression studies, trans-
formed cell identification, and to use as a reporter gene in whole-cell biosensors.
Unfortunately, the requirement of an excitation source complicates the use of
this reporter in the types of hybrid systems considered. However, GFP may be
used in conjunction with luciferase, shifting the emission spectrum from blue-
green bioluminescence (490 nm peak) to green fluorescence (509 nm peak) [82].
The stability and spectral properties of GFP may be modified through struc-
tural alterations of the wild-type protein [25, 57]. Notably, several mutants
with altered excitation and emission spectra have been developed, including
blue-, red-, cyan- and yellow-shifted variants. This spectral diversity provides
potentially important flexibility for cell-to-chip communication by allowing
parallel transmission channels.
Uroporphyrinogen (Urogen) III Methyltransferase
UMT has been identified and purified from several different organisms and
exists in two forms [90]. The first form is encoded by the
cobA
genes in
Bacillusmegaterium
[86],
Methanobacterium ivanovii
[14],
Propionibacterium
freudenreichii
[90], and
Pseudomonas denitrificans
[13], and the second form
is encoded by the
cysG
gene in
E. coli
[113] and
Salmonella typhimurium
[44]. UMT catalyzes the
S
-adenosyl-L-methionine (SAM)-dependent addition
of two methyl groups to the substrate, urogen III. This produces dihydrosiro-
hydrochlorin (precorrin-2), which can be oxidized to a fluorescent product,
sirohydrochlorin, or once again through UMT action accept the addition of a
third methyl group yielding a second fluorescent product, trimethylpyrrocor-
phin. Both products emit an orange-red to red (590-770 nm) fluorescence in
response to UV light excitation at 300 nm [87]. The urogen III substrate can
be found in all organisms [87], allowing UMT to function as a reporter gene
without requiring the addition of a substrate or other cofactors other than the
