Transcript brian_leftright

Need
•Applying Luciferase
and its components in
reaction for making a
bio-detector.
•Determining oxygen
concentrations in
environments
Bioluminescence
C
Lux Operon
D
A
B
E
G
Flavin reductase
Fatty acid reductase
Luciferase
-LuxCDE -fatty acid reductase- converts organic compounds to
oxidizable luciferins
An aldehyde and fmnh2 allow luciferase and luciferin to combine which in its
- AB have been identified as the alpha and beta subunits of luciferase oxidations phase, creates light. Luciferase allows the oxygen to combine with
- LuxG has been found and is shown to be an effective flavin reductase the luciferin which produces photons of light at around 490 nanometers
making a blue green light.
Luciferase
-Vibrio campbellii instead of Vibrio harveyi was used
- (Pimchai, 2008) The superior thermo stability and tighter binding of
FMNH (-) make Vibrio campbellii a more tractable Luciferase
- (Jaffe, 2008) Vibrio campbellii is used which has been shown to be more
stable, stronger and a better light producer than its homologue.
Coupled Assay
Luciferase reaction
+
C1:FMN + NADH
FMNH+NAD +H2O2
Luciferase
FMNH2 + RCHO + O2
FMN + RCOOH + H2O
Light (hv)

C1, a cofactor, combines with FMN which is the flavin mono nucleotide and a substrate. This caused the FMN to be reduced to FMNH2 and produces and oxidized
NAD+ and hydrogen peroxide. The FMNH2, aldehyde and O2 react under the luciferase and produce photons as a by product to FMN, acid and water. C1 provides
enough FMN to the system, however the excess FMNh2 added to the system boost its light producing capabilities. With the increase of free FMNH2 not produced
by the C1:FMN, competition occurs between the two channels because of the Free FMNH2 utilizing more oxygen decreasing the production of NAD+.
Literature Review
- Hastings (1999)- “The Role of Oxygen in
the Photo excited Luminescence of
Bacterial Luciferase”
- Nealson (2002)- “The Total Luminous
Efficiency of Luminous Bacteria”
Purpose
Develop a method of correlating the luminosity of the luciferase
reaction with the oxygen concentration in a given environment
Results
 Standard curves were calculated based on the results of the various test
 Equations of the curves calculated by breaking graphs in to parts and
creating trend lines of either polynomial equations or algorithmic
equations in order to define graphs
 Equations shown are for trials containing no extra FMN added to the
system along with HPA and 9.2 micro molar solution of C1 and Lux
4
-0.000x
3
0.008x
y=
+
2
0.141x + 1.356x + 20.69
R² = 1
Equation of curve with range
less than 146 a.u
3
0.001x
2
0.218x
y=
+ 16.38x 246.4
R² = 1
Equation of curve with range
greater than 146a.u
Discussion
Adding free FMN causes the increase of hydrogen peroxide which
decreases the light production path.
Taking away free FMN created a higher yield of light since the wasteful
FMNH- consumption to produce H2O2 was lessen.
FMNH2 in higher oxygen concentrations reacts with oxygen rather than
aldehyde to produce H2O2 which inhibits FMNH2 from going into the
luciferase reaction
Conclusion
The oxygen concentration of the environment has an impact on the
luminosity of Vibro campbellii luciferase
The luminosity of the reaction can be used to correlate the oxygen
concentration of a given environment
Future 0Studies
-Using increasing amounts of concentrations of Lux in order to obtain a
more defferiantable curve
-Using different strains and species of Lux in order to test for their effect
on the system
Bibliography
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