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.com
Volume 8
Journal of Biotechnology & Biomaterials
ISSN: 2155-952X
Biotech Congress 2018 & Enzymology 2018
March 05-07, 2018
JOINT EVENT
20
th
Global Congress on
Biotechnology
3
rd
International Conference on
Enzymology and Molecular Biology
&
March 05-07, 2018 London, UK
Imaging of vitamin B
12
dynamics by genetically encoded fluorescent nanosensor in living cells
Mohd Mohsin
1
, Mohammad Ahmad
2
and
Altaf Ahmad
3
1
Jamia Millia Islamia, India
2
Jamia Hamdard, India
3
Aligarh Muslim University, India
V
itamin B
12
(cobalamin) is a co-factor of various enzymes and involved in the metabolism of every cell of human body.
Deficiency of vitamin B
12
causes various neurological abnormalities and pernicious anemia. Various methods like isotopic
labeling MS and NMR have been used for measuring the level of cellular metabolites or signaling molecules, but these methods
require the disruption and fractionation of tissues which suffer from contamination. Genetically encoded FRET-based sensors
have been constructed to determine the metabolite concentration in live cells. Here, we report the designing of fluorescence
resonance energy transfer-based nanosensors for direct visualization of changes in cobalamin concentration in intact living
cells. Initially, a construct was designed by using the vitamin B
12
binding protein (BtuF), cyan (CFP) and yellow (YFP) variants
of green fluorescent protein. This construct was then shuttled in different expression vectors. This FRET sensor was named
as SenVitAL (sensor for vitamin anemia linked) which is found to be very specific for vitamin B
12
. This sensor is stable to pH
changes, and measures the vitamin B
12
in a concentration-dependent manner with an apparent affinity of (K
d
) ~157 µM. In
case of
E. coli
, increase in the emission intensity ratio was specifically observed after exposure to vitamin B
12
. Both
in vitro
and
in vivo
measurements, FRET ratio rises after with the addition of vitamin B
12
. Moreover, the results show that the SenVitAL
can evaluate the vitamin B
12
concentration in the cytosol of yeast and mammalian cells, proving its potential in eukaryotic
system. Consequently, the sensor can serve as novel indicator to investigate the vitamin B
12
flux and, would help to elucidate
their complex roles in metabolism.
mohsin4biotech@gmail.comJ Biotechnol Biomater 2018, Volume 8
DOI: 10.4172/2155-952X-C2-092